Project Summary Glycans are known to be involved in including cell growth and differentiation, cell?cell communication, immune response, and intracellular signaling events. Glycans also feature prominently in human disease. In many different human disorders, the cellular glycome is altered. The key unsolved problems in the glycosciences are the tools for study the temporal and spatial distribution of glycans on/in intact cells, which is important to the revelation of glycan-related biological events. We propose to develop a robust, integrated platform for the rapid discovery and characterization of high-affinity nanobodies (i.e., single domain-antibody, sdAbs) against defined glycan structures of biomedical importance. The proposed project seeks to overcome the critical barriers in producing high-affinity glycan specific nanobodies: i) poor immunogenicity of glycans ii) difficulties and labor-intensive nature for immobilization of carbohydrate antigens onto carrier as immunogens. In this Phase I proof-of-concept demonstration, we will select and validate high-affinity nanobodies to tumor-associated glycans antigens of Thomsen-Friedenreich antigen (Tn, Gal(b1-3)GalNAc), the sialyl-Tn (Neu5Ac?2,6GalNAc?1) antigen, and sialyl Lewisx (Neu5Ac?2-3Gal?1-4[Fuc?1-3]GlcNAc?- ). The long-term goal is to buildup libraries of nanobodies to a diversity of glycan structures. Such an effort is anticipated to yield a set of well-characterized new glycan specific affinity reagents that are suitable for probing temporal and spatial expression of glycans in cells and tissues, and provide accessible reagents for `non-glyco' experts in biology and biomedical field.